TGF-beta has been implicated as a critical regulator of the rate of chondrocyte maturation in the post-natal animal. Our preliminary data demonstrates that TGF-beta induces chondrogenesis and inhibits chondrocyte maturation, and activates both Smad and ATF-2 signaling events Finally, our data suggest that Smad3 and ATF-2 act cooperatively to mediate TGF-beta effects in chondrocytes. The current proposal uses a genetic approach, with complementary in vitro and in vivo murine models, to examine the hypothesis that both Smad3 and ATF-2 are critical molecular effectors of TGF-beta effects on chondrogenesis and chondroctye maturation and work cooperatively to mediate TGF-beta effects. Aim I examines the role of Smad3 and ATF-2 as effectors of TGF-beta signaling during chodrogenesis and chondrocyte differentiation using murine cells deficient in Smad3 and ATF-2, alone or in combination. In Aim 2, deletion of ATF-2 is targeted to cartilage through the development of a coI2A-CRE;ATF-2 loxP mouse. The ATF-2 -/- mouse dies at birth of pulmonary disease, but it is our hypothesis that the conditional mutation will have premature chondrocyte maturation post natal and will develop progressively abnormal growth and joint function, as observed in Smad3 -/- mice. Furthermore, we hypothesize that crossing these animals with Smad3 -/- mice will result in a phenotype more severe than that observed in animals with a single gene deletion. In Aim 3 we examine the role of Smad3 and ATF-2 on skeletal repair, using murine in vivo ectopic endochondral bone formation and fracture healing models. These studies will examine the hypothesis that the TGF-beta signaling molecules Smad3 and ATF-2 are essential for normal endochondral bone repair in the post natal animal. We further hypothesize that combined deletion of Smad3 and ATF-2 will result in a more severe phenotype with more profound abnormalities in endochondral bone repair. Thus, the experiments use a genetic approach to examine the role of TGF-beta signaling on skeletal growth and development, osteoarthritis, and tissue repair and have great potential to define important molecular targets that can directly benefit orthopaedic patients.